Inhibition of mitochondrial oxidative phosphorylation progresses to uncoupling when opening of cyclosporin A-sensitive permeability transition pores increases permeability of the mitochondrial inner membrane to small solutes. Involvement of the mitochondrial permeability transition (MPT) in necrotic and apoptotic cell death is implicated by demonstrations of protection by cyclosporin A against oxidative stress, ischemia/reperfusion, tumor necrosis factor-alpha exposure, Fas ligation, calcium overload, and a variety of toxic chemicals. Confocal microscopy directly visualizes the MPT in single mitochondria within living cells from the translocation of impermeant fluorophores, such as calcein, across the inner membrane. Simultaneously, mitochondria release potential-indicating fluorophores. Subsequently, mitochondria swell, causing outer membrane rupture and release of cytochrome c and other proapoptotic proteins from the intermembrane space. In situ a sequence of decreased NAD(P)H, increased free calcium, and increased reactive oxygen species formation within mitochondria promotes the MPT and subsequent cell death. Necrotic and apoptotic cell death after the MPT depends, in part, on ATP levels. If ATP levels fall profoundly, glycine-sensitive plasma membrane permeabilization and rupture ensue. If ATP levels are partially maintained, apoptosis follows the MPT. The MPT also signals mitochondrial autophagy, a process that may be important in removing damaged mitochondria. Cellular features of necrosis, apoptosis, and autophagy frequently occur together after death signals and toxic stresses. A new term, necrapoptosis, describes such death processes that begin with a common stress or death signal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmed cellular resorption (apoptosis), depending on modifying factors such as ATP.

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101- amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1-SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.

Mycobacterium tuberculosis is able to persist in the human host for decades in an apparently dormant state where it is presumed to reside in an hypoxic environment. This can be mimicked by the Wayne culture model in which progressive oxygen depletion causes the bacteria to shift into a non-replicating state. We investigated global gene expression in aerobic (roller), microaerophilic (NRP1) and anaerobic (NRP2) cultures. A number of genes were significantly up-regulated as compared to aerobic culture; 178 in NRP1, 210 in NRP2, 88 in both. The two states showed distinct gene expression profiles, although a number of membrane and transmembrane proteins were induced in both conditions. A number of regulatory proteins were up-regulated in NRP2. Glycine dehydrogenase, nitrate reductase and alpha-crystallin were induced in both stages, as were fatty acid metabolism genes including fadD26 and mas and genes of the DosR regulon. In a comparison with other stress conditions, there were more similarities between anaerobic conditions and carbon starvation or heat shock than between microaerophilic conditions and carbon starvation or heat shock, but as expected microaerophilic and anaerobic conditions showed the most similar profile. Our results indicate that a large number of genes are up-regulated during the shift into the persistent state.

The integrin alpha(9)beta(1) mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH(2)-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for alpha(9)beta(1). In this report, we used site-directed mutagenesis and synthetic peptides to identify the alpha(9)beta(1) recognition sequence in osteopontin. alpha(9)-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH(2)-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-alpha(9) monoclonal antibody Y9A2, indicating the presence of a non-RGD alpha(9)beta(1) recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on alpha(9)beta(1)-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr(165), blocked alpha(9)beta(1)-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin beta(1) subunit on alpha(9)-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to alpha(9)beta(1) and is responsible for alpha(9)beta(1)-mediated cell adhesion to the NH(2)-terminal fragment of osteopontin.

Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.

Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.

Activation of the NMDA subtype of ionotropic glutamate receptors requires binding of both L-glutamate and the coagonist glycine. Site-directed mutagenesis of the NMDAR1 (NR1) subunit revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of glycine binding. Glutamate efficacy was little affected by mutations at these positions; however, inhibition of channel gating by the glycine antagonist 7-chlorokynurenic acid was drastically reduced. In addition, glutamine (Q387), valine (V666), and serine (S669) substitutions were found to reduce glycine efficacy. Since the mutated residues correspond to positions forming the binding site of homologous bacterial amino acid-binding proteins, a common amino acid-binding fold appears to be conserved from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain.

The unimolecular and low energy collision-induced fragmentation reactions of the MH(+) ions of N-acetyl-tri-alanine, N-acetyl-tri-alanine methyl ester, N-acetyl-tetra-alanine, tetra-alanine, penta-alanine, hexa-glycine, and Leu-enkephalin have been studied with a particular emphasis on the formation and fragmentation of B n (n=3,4,5) ions. In addition, the metastable ion fragmentation reactions of protonated tetra-glycine, penta-glycine, and Leu-enkephalin amide have been studied. B n ions are prominent stable species in all spectra. The B n ions fragment, in part, by elimination of CO to form A n ions; this reaction occurs on the metastable ion time scale with a substantial release of kinetic energy (T 1/2=0. 3-0. 5 eV) that indicates that a stable configuration of the B n ion fragments by way of a reacting configuration that is higher in energy than the fragmentation products, A n + CO. Ab initio calculations strongly suggest that the stable configuration of the B3 and B4 ions is a protonated oxazolone formed by interaction of the developing charge with the next-nearest carbonyl group as HX is lost from the protonated species H-(Yyy) n -X · H(+). The higher B n ions also fragment, in part, to form the next-lower B ion, presumably in its stable protonated oxazolone form. This reaction is rationalized in terms of the three-dimensional structure of the B n ions and it is proposed that the neutral eliminated is an α-lactam.

We have determined the size and sequence of 8 exons in the gene that specifies chick type 1 alpha 2 collagen. These 8 exons represent three different segments of the gene, but all encode information for the helical portion of the protein. Seven exons have a length of 54 bp, and the 8th has a size of 99 bp. The sequences within these exons vary except for th glycine codons, which occur every third triplet. Each exon begins with a glycine codon and ends with a triplet that precedes a glycine codon. The size and the sequences of the introns do not show any homology except at their ends. Of 7 introns examined the first six bases at the 5' end of 5 of these are identical. The sequences at the 3' end of the introns also show homologies. Our results imply that the ancestral gene for collagen arose by multiple duplications of a single genetic unit containing a 54 bp condig segment. The sequences within these exons drifted by successive point mutations and in some cases by additions or deletions of 9 bp or multiples of 9 bp.

Previously we demonstrated the expression of a plant embryo-specific gene encoding the alpha' subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), in transgenic petunia plants. To examine the regulatory elements that control the expression of this embryo-specific gene (Gmg17.1), a series of deletion mutants was made that contain the alpha'-subunit gene flanked in the 5' direction from +14 nucleotides to -8.5 kilobases (kb) relative to the site of transcription initiation. Each of these deletion mutants was introduced into the genome of petunia cells with the help of Ti-plasmid-derived vectors. Petunia plants were regenerated from transformed cells and expression of the introduced soybean gene was examined. When the alpha'-subunit gene was flanked by 159 nucleotides upstream (Gmg17.1 delta-159), the gene was expressed at a low level in immature embryos. When the gene was flanked by 257 nucleotides upstream of the site of transcription initiation (Gmg17.1 delta-257), a high level of expression was obtained. An additional 8 kb of DNA sequence (which includes the sequence GTGGATAG at -560, which is identical to the core enhancer sequence of simian virus 40 and some animal genes) did not significantly increase the level of expression. The increase in expression level between the delta-159 and delta-257 mutants was at least 20-fold. Analysis of the nucleotides between delta-159 and delta-257 reveals four repeats of a 6-base-pair (G + C)-rich sequence (see formula in text). The deletion Gmg17.1 delta-159 contains a single AACCCA sequence. We suggest that the (G + C)-rich repeats play a critical role in determining the level of expression of the transgenic plants.

We have characterized the expression of two members of a class of Arabidopsis thaliana glycine-rich, putative RNA-binding proteins that we denote Ccr1 and Ccr2. Southern blot analysis indicates that Ccr1 and Ccr2 are members of a small gene family. Both Ccr1 and Ccr2 mRNA levels were influenced by a circadian rhythm that has an unusual phase for plants, with maximal accumulation at 6:00 PM and minimal accumulation at 10:00 AM. The level of CCR1 protein, however, remained relatively constant throughout the cycle. The transcript accumulation patterns of the Ccr1 and Ccr2 genes differed considerably from conditions that affect the expression of similar genes from maize, sorghum, and carrot. Levels of Ccr1 and Ccr2 mRNAs were unchanged in wounded plants, increased at least 4-fold in cold-stressed plants, and decreased 2- to 3-fold in abscisic acid-treated plants. Ccr1 transcript levels decreased in response to drought, whereas Ccr2 transcript levels increased under the same conditions. Based on the presence of additional Ccr transcripts in dark-grown plants, we propose that Ccr transcripts may be subjected to a light- or dark-mediated regulation.

The infectible cells of soybean roots appear to be located at any given time just above the zone of root elongation and just below the position of the smallest emergent root hairs. The location of infectible cells on the primary root at the time of inoculation was inferred from the position of subsequent nodule development, correcting for displacement of epidermal cells due to root elongation. Marks were made on the seedling growth pouches at the time of inoculation to indicate the position of the root tip and the zones of root hair development. Virtually all of the seedlings developed nodules on the primary root above the marks made at the root tips at the time of inoculation. None of the plants formed nodules on the root where mature root hairs were present at the time of inoculation. These results and profiles of nodulation frequency indicate that the location of infectible cells is developmentally restricted. When inoculations were delayed for intervals of 1 to 4 hours after marking the positions of the root tips, progressively fewer nodules were formed above the root tip marks, and the uppermost of these nodules were formed at progressively shorter distances above the marks. These results indicate that the infectibility of given host cells is a transient property that appears and then is lost within a few hours. The results also indicate that host responses leading to infection and nodulation are triggered or initiated in less than 2 hours after inoculation. The extent of nodulation above the root tip mark increased in proportion to the logarithm of the number of bacteria in the inoculum.

The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance 13C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (less than 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.

Epstein-Barr virus (EBV)-induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.

The formation of dendritic spines during development and their structural plasticity in the adult brain are critical aspects of synaptogenesis and synaptic plasticity. Many different factors and proteins have been shown to control dendritic spine development and remodeling (Ethell and Pasquale, 2005). The extracellular matrix (ECM) components and their cell surface receptors, integrins, have been found in the vicinity of synapses and shown to regulate synaptic efficacy and play an important role in long-term potentiation (Bahr et al., 1997; Chavis and Westbrook, 2001; Chan et al., 2003; Lin et al., 2003; Bernard-Trifilo et al., 2005). Although molecular mechanisms by which integrins affect synaptic efficacy have begun to emerge, their role in structural plasticity is poorly understood. Here, we show that integrins are involved in spine remodeling in cultured hippocampal neurons. The treatment of 14 d in vitro hippocampal neurons with arginine-glycine-aspartate (RGD)-containing peptide, an established integrin ligand, induced elongation of existing dendritic spines and promoted formation of new filopodia. These effects were also accompanied by integrin-dependent actin reorganization and synapse remodeling, which were partially inhibited by function-blocking antibodies against beta1 and beta3 integrins. This actin reorganization was blocked with the NMDA receptor (NMDAR) antagonist MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide) also suppressed RGD-induced actin reorganization and synapse remodeling. Our findings show that integrins control ECM-mediated spine remodeling in hippocampal neurons through NMDAR/CaMKII-dependent actin reorganization.

The responses to excitatory and inhibitory amino acids and peptides were investigated in isolated rat spinal dorsal horn neurons (laminae I-V) of young rats using the whole-cell voltage-clamp technique. The treatment of spinal slices with low concentrations of enzymes and mechanical dissociation yielded isolated neurons that were sensitive to excitatory amino acids (glutamate, kainate, quisqualate and N-methyl-D-aspartate (NMDA), inhibitory amino acids (gamma-aminobutyric acid (GABA), glycine) and peptides (substance P, calcitonin gene-related peptide (CGRP). The responses of dorsal horn neurons to NMDA were potentiated by glycine and CGRP, whereas GABAA responses were enhanced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Our observations indicate that there is reasonable agreement between many of the responses of isolated neurons and those studied in in vivo and in vitro slice and culture preparations.

BACKGROUND

The Cation Diffusion Facilitator (CDF) family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters.

RESULTS

Representative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes) were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed.

CONCLUSIONS

In the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be used to identify new hypothetical CDF members.

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet-triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (N) separating donor and acceptor and show a maximum for the shortest peptides (N = 3) with a time constant (tau = 1/k) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.

To determine whether the oscillating clinical response to levodopa in Parkinson's disease (the "on-off" phenomenon) reflects fluctuations in absorption and transport of the drug, we investigated this phenomenon in nine patients with an oscillating motor state. We studied the response to continuous infusion of levodopa and the effects of meals on the plasma levodopa concentrations and on the clinical response during oral and intravenous administration of the drug. Meals reduced peak plasma levodopa concentrations by 29 per cent and delayed absorption by 34 minutes. Bypassing absorption by constant infusion of the drug produced a stable clinical state lasting for 12 hours in all of six patients and for up to 36 hours in some. High-protein meals or oral phenylalanine, leucine, or isoleucine (100 mg per kilogram of body weight) reversed the therapeutic effect of infused levodopa without reducing plasma levodopa concentrations. Glycine and lysine at identical doses had no effect. We conclude that interference with absorption of levodopa by food and by competition between large neutral amino acids and levodopa for transport from plasma to the brain may be partly responsible for the fluctuating clinical response in patients with Parkinson's disease.

Because of its protein-denaturing ability, urea has played a pivotal role in the experimental and conceptual understanding of protein folding and unfolding. The measure of urea's ability to force a protein to unfold is given by the m value, an experimental quantity giving the free energy change for unfolding per molar urea. With the aid of Tanford's transfer model [Tanford C (1964) J Am Chem Soc 86:2050-2059], we use newly obtained group transfer free energies (GTFEs) of protein side-chain and backbone units from water to 1 M urea to account for the m value of urea, and the method reveals the anatomy of protein denaturation in terms of residue-level free energy contributions of groups newly exposed on denaturation. The GTFEs were obtained by accounting for solubility and activity coefficient ratios accompanying the transfer of glycine from water to 1 M urea. Contrary to the opinions of some researchers, the GTFEs show that urea does not denature proteins through favorable interactions with nonpolar side chains; what drives urea-induced protein unfolding is the large favorable interaction of urea with the peptide backbone. Although the m value is said to be proportional to surface area newly exposed on denaturation, only approximately 25% of the area favorably contributes to unfolding (because of newly exposed backbone units), with approximately 75% modestly opposing urea-induced denaturation (originating from side-chain exposure). Use of the transfer model and newly determined GTFEs achieves the long-sought goal of predicting urea-dependent cooperative protein unfolding energetics at the level of individual amino acid residues.

Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity. Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana ( approximately 0.8 %).

The threonine-glycine (Thr-Gly) encoding repeat within the clock gene period of Drosophila melanogaster is polymorphic in length. The two major variants (Thr-Gly)17 and (Thr-Gly)20 are distributed as a highly significant latitudinal cline in Europe and North Africa. Thr-Gly length variation from both wild-caught and transgenic individuals is related to the flies' ability to maintain a circadian period at different temperatures. This phenomenon provides a selective explanation for the geographical distribution of Thr-Gly lengths and gives a rare glimpse of the interplay between molecular polymorphism, behavior, population biology, and natural selection.

Polyploidy is generally not tolerated in animals, but is widespread in plant genomes and may result in extensive genetic redundancy. The fate of duplicated genes is poorly understood, both functionally and evolutionarily. Soybean (Glycine max L.) has undergone two separate polyploidy events (13 and 59 million years ago) that have resulted in 75% of its genes being present in multiple copies. It therefore constitutes a good model to study the impact of whole-genome duplication on gene expression. Using RNA-seq, we tested the functional fate of a set of approximately 18 000 duplicated genes. Across seven tissues tested, approximately 50% of paralogs were differentially expressed and thus had undergone expression sub-functionalization. Based on gene ontology and expression data, our analysis also revealed that only a small proportion of the duplicated genes have been neo-functionalized or non-functionalized. In addition, duplicated genes were often found in collinear blocks, and several blocks of duplicated genes were co-regulated, suggesting some type of epigenetic or positional regulation. We also found that transcription factors and ribosomal protein genes were differentially expressed in many tissues, suggesting that the main consequence of polyploidy in soybean may be at the regulatory level.